Recently, significant effort has been invested in developing and improving recombinant poxvirus-based technologies. Poxvirus-based vectors have been identified as beneficial for a number of uses such as generating immune responses through vaccines, in the development of new vaccine therapies, and use in gene therapy applications. The advantages associated with recombinant poxvirus vectors are well-known and include efficient delivery of genetic material to multiple cell types; generous levels of protein expression; and the ability to elicit cell-mediated immune responses in addition to antibody-based responses.
Poxviruses are well-known cytoplasmic viruses, thus any genetic material carried by such viral vectors normally remains in the cytoplasm without the disadvantage of inadvertent integration into the host cell genome. Poxviruses can be readily genetically engineered to contain and express foreign genes that have been inserted into their genome using, for example, cloning techniques such as homologous recombination. These foreign genes can encode a wide range of proteins, such as antigens that induce protection against one or more infectious agents, immune modulating proteins such as co-stimulatory molecules, or enzymatic proteins. For example, recombinant poxviruses have been engineered to express immunizing antigens of herpes virus, influenza, and human immunodeficiency virus (HIV).
One of the main advantages of poxviruses as vectors is their large genome size, which permits the insertion of a large amount of heterologous genetic material including, for example, multiple genes (i.e., as a multivalent vector). However, the heterologous genetic material must be inserted at an appropriate site within the pox genome for the recombinant virus to remain viable. Thus, the genetic material must be inserted at a site in the viral DNA which is non-essential.
A well-established approach for the cloning of recombinant poxviruses is based on two separate recombination events. During the first recombination step, a gene of interest and a reporter and/or marker cassette are integrated into a viral genome. For the selection process, an antibiotic resistance gene is commonly used. Following the subsequent isolation of recombinant poxviruses from the pool of recombinant and non-recombinant poxviruses using the selection/marker cassette, the selection and marker cassette should be deleted if the recombinant poxvirus is intended for use as, for example, a vaccine in humans. For this purpose, a second recombination event must be performed involving further passaging and plaque purification of the recombinant poxvirus. Consequently, presently known techniques for cloning recombinant poxviruses are usually time-consuming and laborious endeavors, especially when compared to those procedures commonly used for the cloning of other types of recombinant expression vectors.
Accordingly, there is a need in the art for improved cloning systems and methods for the efficient generation of recombinant poxviruses.